The blood-brain barrier (BBB) is composed of endothelial cells, pericytes and astrocytes [1], and serves as interface between the blood flow and the central nervous system (CNS). Since BBB deregulation plays important roles in the pathogenesis of several CNS diseases spanning from brain tumors to stroke, understanding the molecular mechanisms controlling BBB functions might help to unveil new therapeutic targets to brain pathologies. Extracellular nucleotides are important signaling molecules both in physiological and pathological conditions. Their actions are mediated by 7 ionotropic P2X and 8 metabotropic P2Y purinergic receptors, and terminated by metabolizing enzymes, named ectonucleotidases (NTPDases) and 5’-nucleotidase [2]. To date, the role of purinergic transmission in controlling BBB functions is not fully understood yet. Therefore, we used a new in vitro cell culture model of BBB [3] to investigate the expression and distribution of NTPDases and P2Y receptors either in control conditions or following exposure to ischemia. Semi quantitative RT-PCR analysis showed that astrocytes and pericytes expressed all the cloned P2Y receptors, however endothelial cells showed the presence only the P2Y1,2,4 subtypes. NTPDase1 and 2 enzymes were expressed at different level but by all the three types of cell. Application of oxygen-glucose deprivation (OGD), which mimics cytotoxicity induced by ischemia in vivo, showed extremely high susceptibility to cell death of endothelial cells, whereas astrocytes and pericytes were more resistant. Semi-quantitative densitometry assay highlighted increased ecto-ATPase activity following exposure to OGD in the three types of cell population, either they were grown separately or in triple co-culture. Our data show the usefulness of this new in vitro model to demonstrate a role for extracellular nucleotides in modulating BBB responses to ischemic events, and to determine if the purinergic system could represent a new target for the development of effective therapies to brain pathologies. Figure 1: Table of contents. (A) ATP hydrolyzing enzymes and some of the pharmacological targets of ATP metabolites. (B) schematic representation of the blood brain barrier in vivo and of its reproduction in vitro. (C) experimental plan. Figure 2: Schematic drawing of the BBB model and its electron microscopic picture. (A) Endothelial cells (green), pericytes (brown) and astrocytes (blue) as they are located in the well of Transwell cell culture plate. (B) Electron micrograph: single layer of endothelial cells on the top and multilayer of pericytes on the bottom of the 400 nm thick membrane. References [1] N. Abbott, L. Rönnbäck, E. Hansson, Astrocyte-endothelial interactions at the blood-brain barrier. Nat Rev Neurosci, 7, 41-53, 2006. [2] G. Yegutkin, Nucleotide- and nucleoside-converting ectoenzymes: Important modulators of purinergic signalling cascade. Biochim Biophys Acta, 1783, 673-694, 2008. [3] S. Nakagawa, M. Deli, H. Kawaguchi, T. Shimizudani, T. Shimono, A. Kittel, K. Tanaka, M. Niwa, A new blood-brain barrier model using primary rat brain endothelial cells, pericytes and astrocytes. Neurochem Int, 54, 253-263, 2009.

BBB and the purinergic system / G. Magni. ((Intervento presentato al convegno Hungarian Microscopy Conference tenutosi a Siófok nel 2010.

BBB and the purinergic system

G. Magni
2010

Abstract

The blood-brain barrier (BBB) is composed of endothelial cells, pericytes and astrocytes [1], and serves as interface between the blood flow and the central nervous system (CNS). Since BBB deregulation plays important roles in the pathogenesis of several CNS diseases spanning from brain tumors to stroke, understanding the molecular mechanisms controlling BBB functions might help to unveil new therapeutic targets to brain pathologies. Extracellular nucleotides are important signaling molecules both in physiological and pathological conditions. Their actions are mediated by 7 ionotropic P2X and 8 metabotropic P2Y purinergic receptors, and terminated by metabolizing enzymes, named ectonucleotidases (NTPDases) and 5’-nucleotidase [2]. To date, the role of purinergic transmission in controlling BBB functions is not fully understood yet. Therefore, we used a new in vitro cell culture model of BBB [3] to investigate the expression and distribution of NTPDases and P2Y receptors either in control conditions or following exposure to ischemia. Semi quantitative RT-PCR analysis showed that astrocytes and pericytes expressed all the cloned P2Y receptors, however endothelial cells showed the presence only the P2Y1,2,4 subtypes. NTPDase1 and 2 enzymes were expressed at different level but by all the three types of cell. Application of oxygen-glucose deprivation (OGD), which mimics cytotoxicity induced by ischemia in vivo, showed extremely high susceptibility to cell death of endothelial cells, whereas astrocytes and pericytes were more resistant. Semi-quantitative densitometry assay highlighted increased ecto-ATPase activity following exposure to OGD in the three types of cell population, either they were grown separately or in triple co-culture. Our data show the usefulness of this new in vitro model to demonstrate a role for extracellular nucleotides in modulating BBB responses to ischemic events, and to determine if the purinergic system could represent a new target for the development of effective therapies to brain pathologies. Figure 1: Table of contents. (A) ATP hydrolyzing enzymes and some of the pharmacological targets of ATP metabolites. (B) schematic representation of the blood brain barrier in vivo and of its reproduction in vitro. (C) experimental plan. Figure 2: Schematic drawing of the BBB model and its electron microscopic picture. (A) Endothelial cells (green), pericytes (brown) and astrocytes (blue) as they are located in the well of Transwell cell culture plate. (B) Electron micrograph: single layer of endothelial cells on the top and multilayer of pericytes on the bottom of the 400 nm thick membrane. References [1] N. Abbott, L. Rönnbäck, E. Hansson, Astrocyte-endothelial interactions at the blood-brain barrier. Nat Rev Neurosci, 7, 41-53, 2006. [2] G. Yegutkin, Nucleotide- and nucleoside-converting ectoenzymes: Important modulators of purinergic signalling cascade. Biochim Biophys Acta, 1783, 673-694, 2008. [3] S. Nakagawa, M. Deli, H. Kawaguchi, T. Shimizudani, T. Shimono, A. Kittel, K. Tanaka, M. Niwa, A new blood-brain barrier model using primary rat brain endothelial cells, pericytes and astrocytes. Neurochem Int, 54, 253-263, 2009.
2010
Settore BIO/14 - Farmacologia
BBB and the purinergic system / G. Magni. ((Intervento presentato al convegno Hungarian Microscopy Conference tenutosi a Siófok nel 2010.
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/2434/239037
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